| 1. |
- Aisenbrey, Christopher, et al.
(författare)
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How is protein aggregation in amyloidogenic diseases modulated by biological membranes?
- 2008
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Ingår i: European Biophysics Journal. - : SpringerLink. - 0175-7571 .- 1432-1017. ; 37:3, s. 247-55
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Tidskriftsartikel (refereegranskat)abstract
- The fate of proteins with amyloidogenic properties depends critically on their immediate biochemical environment. However, the role of biological interfaces such as membrane surfaces, as promoters of pathological aggregation of amyloidogenic proteins, is rarely studied and only established for the amyloid-β protein (Aβ) involved in Alzheimer’s disease, and α-synuclein in Parkinsonism. The occurrence of binding and misfolding of these proteins on membrane surfaces, is poorly understood, not at least due to the two-dimensional character of this event. Clearly, the nature of the folding pathway for Aβ protein adsorbed upon two-dimensional aggregation templates, must be fundamentally different from the three-dimensional situation in solution. Here, we summarize the current research and focus on the function of membrane interfaces as aggregation templates for amyloidogenic proteins (and even prionic ones). One major aspect will be the relationship between membrane properties and protein association and the consequences for amyloidogenic products. The other focus will be on a general understanding of protein folding pathways on two-dimensional templates on a molecular level. Finally, we will demonstrate the potential importance of membrane-mediated aggregation for non-amphiphatic soluble amyloidogenic proteins, by using the SOD1 protein involved in the amyotrophic lateral sclerosis syndrome.
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| 2. |
- Byström, Roberth, 1971-, et al.
(författare)
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Disordered proteins : Biological membranes as two-dimensional aggregation matrices
- 2008
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Ingår i: Cell Biochemistry and Biophysics. - : Springer Science and Business Media LLC. - 1085-9195 .- 1559-0283. ; 52:3, s. 175-189
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Forskningsöversikt (refereegranskat)abstract
- Aberrant folded proteins and peptides are hallmarks of amyloidogenic diseases. However, the molecular processes that cause these proteins to adopt non-native structures in vivo and become cytotoxic are still largely unknown, despite intense efforts to establish a general molecular description of their behavior. Clearly, the fate of these proteins is ultimately linked to their immediate biochemical environment in vivo. In this review, we focus on the role of biological membranes, reactive interfaces that not only affect the conformational stability of amyloidogenic proteins, but also their aggregation rates and, probably, their toxicity. We first provide an overview of recent work, starting with findings regarding the amphiphatic amyloid-β protein (Aβ), which give evidence that membranes can directly promote aggregation, and that the effectiveness in this process can be related to the presence of specific neuronal ganglioside lipids. In addition, we discuss the implications of recent research (medin as an detailed example) regarding putative roles of membranes in the misfolding behavior of soluble, non-amphiphatic proteins, which are attracting increasing interest. The potential role of membranes in exerting the toxic action of misfolded proteins will also be highlighted in a molecular context. In this review, we discuss novel NMR-based approaches for exploring membrane–protein interactions, and findings obtained using them, which we use to develop a molecular concept to describe membrane-mediated protein misfolding as a quasi-two-dimensional process rather than a three-dimensional event in a biochemical environment. The aim of the review is to provide researchers with a general understanding of the involvement of membranes in folding/misfolding processes in vivo, which might be quite universal and important for future research concerning amyloidogenic and misfolding proteins, and possible ways to prevent their toxic actions.
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| 3. |
- Khemtémourian, Lucie, et al.
(författare)
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Synthesis and secondary structure in membranes of the Bcl-2 anti-apoptotic domain BH4
- 2006
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Ingår i: Journal of Peptide Science. - : Wiley. - 1075-2617 .- 1099-1387. ; 12, s. 58-64
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Tidskriftsartikel (refereegranskat)abstract
- Solid phase synthesis of BH4, the 26 amino-acid domain (6RTGYDNREIVMKYIHYKLSQRGYEWD31) of the anti-apoptotic Bcl-2 protein has been accomplished using Fmoc chemistry. The use of peculiar cleavage conditions provided high yields after purification such that tens to hundreds of mg could be obtained. A 15N-labelled version of the peptide could also be synthesized for NMR studies in membranes. The peptide purity was not lower than 98% as controlled by UV and MALDI-TOF mass spectrometry. The secondary structure was determined in water, trifluoroethanol (TFE) and in lipid membrane using UV circular dichroism. The peptide shows dominant -sheeted structures in water that convert progressively into -helical features upon addition of TFE or membrane. The amphipathic character of the helix suggests that the peptide might have a structure akin to those of antimicrobial peptides upon interaction with membranes. Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd.
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| 4. |
- Sani, Marc-Antoine, 1979-
(författare)
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Apoptosis Regulation via the Mitochondrial Pathway : Membrane Response upon Apoptotic Stimuli
- 2008
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- The aim of this thesis was the investigation of the mitochondrial response mechanisms upon apoptotic stimuli. The specific objectives were the biophysical characterization of membrane dynamics and the specific roles of lipids in the context of apoptotic regulation occurring at the mitochondrion and its complex membrane systems.The BH4 domain is an anti-apoptotic specific domain of the Bcl-2 protein. Solid phase peptide synthesis was used to produce large amount of the peptide for biophysical studies. A protocol has been established and optimized, guarantying the required purity for biophysical studies. In detail the purification by high performance liquid chromatography and the characterisation via mass spectroscopy are described. The secondary structure of BH4 changes significantly in the presence of lipid vesicles as observed by infrared spectroscopy and circular dichroism. The BH4 peptide aggregates at the membrane surface and inserts slightly into the hydrophobic part of the membrane. Using nuclear magnetic resonance (NMR) and calorimetry techniques, it could even be shown that the BH4 domain modifies the dynamic and organization of the liposomes which mimic a mitochondrial surface. The second study was on the first helix of the pro-apoptotic protein Bax. This sequence called Bax-α1 has the function to address the cytosolic Bax protein to the mitochondrial membrane upon activation. Once again a protocol has been established for the synthesis and purification of this peptide. The aim was to elucidate the key role of cardiolipin, a mitochondria-specific phospholipid, in the interaction of Bax-α1 with the mitochondrial membrane system. The NMR and circular dichroism studies showed that Bax-α1 interacts with the membrane models only if they contain the cardiolipin, producing a strong electrostatic lock effect which is located at the membrane surface.Finally, a new NMR approach was developed which allows the investigation of the lipid response of isolated active mitochondria upon the presence of apoptotic stimuli. The goal was there to directly monitor lipid specific the occurring changes during these physiological activities.
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| 5. |
- Sani, Marc-Antoine, et al.
(författare)
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How does the Bax-α1 targeting sequence interact with mitochondrial membrane? : The role of cardiolipin
- 2009
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Ingår i: Biochimica et Biophysica Acta - Biomembranes. - : Elsevier B.V.. - 0005-2736 .- 1879-2642. ; 1788:3, s. 623-631
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Tidskriftsartikel (refereegranskat)abstract
- A key event in programmed cell death is the translocation of the apoptotic Bax protein from the cytosol towards mitochondria. The first helix localized at the N-terminus of Bax (Bax-α1) can act here as an addressing sequence, which directs activated Bax towards the mitochondrial surface. Solid state NMR (nuclear magnetic resonance), CD (circular dichroism) and ATR (attenuated total reflection) spectroscopy were used to elucidate this recognition process of a mitochondrial membrane system by Bax-α1. Two potential target membranes were studied, with the outer mitochondrial membrane (OM) mimicked by neutral phospholipids, while mitochondrial contact sites (CS) contained additional anionic cardiolipin. 1H and 31P magic angle spinning (MAS) NMR revealed Bax-α1 induced pronounced perturbations in the lipid headgroup region only in presence of cardiolipin. Bax-α1 could not insert into CS membranes but at elevated concentrations it inserted into the hydrophobic core of cardiolipin-free OM vesicles, thereby adopting β-sheet-like features, as confirmed by ATR. CD studies revealed, that the cardiolipin mediated electrostatic locking of Bax-α1 at the CS membrane surface promotes conformational change into an α-helical state; a process which seems to be necessary to induce further conformational transition events in activated Bax which finally causes irreversible membrane permeabilization during the mitochondrial apoptosis.
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| 6. |
- Sani, Marc-Antoine, et al.
(författare)
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Magic-angle phosphorus NMR of functional mitochondria : in situ monitoring of lipid response under apoptotic-like stress
- 2009
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Ingår i: The FASEB Journal. - : The Federation of American Societies for Experimental Biology. - 0892-6638 .- 1530-6860. ; 23:9, s. 2872-2878
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Tidskriftsartikel (refereegranskat)abstract
- Using a noninvasive, solid-state magic-angle spinning nuclearmagnetic resonance (MAS NMR) approach, we track ex vivo thebehavior of individual membrane components in isolated, activemitochondria (model system: potato tubers) during physiologicalprocesses. The individual phosphatidylcholine (PC), phosphatidylethanolamine(PE), and cardiolipin (CL) membrane constituents were identifiedas distinct lines by applying MAS 31P NMR on extracted lipidmembranes. However, the CL NMR signal appeared to be very broadin functional mitochondria, indicating a tight complex formationwith membrane protein. Calcium stress induced severe membranedegradation without recovery of a single CL NMR resonance. Thissuggests that calcium overload destroys the outer mitochondrialmembrane and does not modify strongly the CL protein complexesin the inner membrane; a conclusion confirmed by respiratorycontrols. Conversely, mitochondrial membrane disruption on timedegradation or mechanical stress generates clearly visible identicalCL NMR signals, similar to those observed in rehydrated lipidextracts. Similarly, noninvasive based NMR tracking of lipidsin response to diverse physiological stimuli can easily be usedfor other organelles and whole living cells. Sani, M.-A., Keech,O., Gardeström, P., Dufourc, E. J., Gröbner, G. Magic-anglephosphorus NMR of functional mitochondria: in situ monitoringof lipid response under apoptotic-like stress.
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| 7. |
- Sani, Marc-Antoine, et al.
(författare)
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Pro-apoptotic bax-α1 synthesis and evidence for β-sheet to α-helix conformational change as triggered by negatively charged lipid mβembranes
- 2007
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Ingår i: Journal of Peptide Science. - : Wiley. - 1075-2617 .- 1099-1387. ; 13:2, s. 100-106
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Tidskriftsartikel (refereegranskat)abstract
- Solid phase synthesis of Bax-α1, the 25 amino acids domain (14TSSEQIMKTGALLLQGFIQDRAGRM38) of the pro-apoptotic Bax protein has been accomplished using Fmoc chemistry. A new fast and harmless protocol is described for complete TFA removal from the purified peptide powder leading to a final purity greater than 98% as controlled by 19F-NMR, UV and MALDI-TOF mass spectrometry. Secondary structure was determined in various solution and membrane media using UV Circular Dichroism. In water solution, Bax-α1 is present as a mixture of β-sheet and unstructured (random coil) conformations. A marked change from β-sheet to α-helix secondary structures is observed upon interaction with negatively charged phospholipids vesicles whereas neutral lipid membranes have no significant effect on the aqueous peptide conformation. Results are discussed in terms of Bax binding to mitochondrial membranes.
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| 8. |
- Sani, Marc-Antoine, et al.
(författare)
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Restriction of lipid motion in membranes triggered by β-sheet aggregation of the anti-apoptotic BH4 domain
- 2008
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Ingår i: The FEBS Journal. - : FEBS Press. - 1742-464X .- 1742-4658. ; 275:3, s. 561-572
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Tidskriftsartikel (refereegranskat)abstract
- The regulative BH4 domain of human Bcl-2 protein exerts its anti-apoptic activity via the mitochondrion. In the present study, we investigated the molecular interactions of this domain with negatively charged liposomes mimicking the outer mitochondrial membrane. To model the overproduction of Bcl-2 found in cancer processes, we studied the impact of elevated concentrations of its regulative BH4 segment on these mitochondrial membranes from the peptide and lipid perspective. Combined solid state 2H-NMR and differential scanning calorimetry revealed the coexistence of small sized fluid and rigid membrane domains over a large temperature range, which is confirmed by 31P-NMR at 30 °C. The latter are stabilized, in a cholesterol-like manner, by the presence of a BH4 peptide. In the same time scale, the reduction of the headgroup order is seen in the static 14N and 31P-NMR spectra when BH4 inserts into the bilayers. Indeed, attenuated total reflection spectroscopy indicated a dominant aggregated β-sheet secondary structure of BH4 with a 42° tilt relative to the membrane surface. These results are discussed in terms of membrane stabilization versus apoptotic mechanisms at the outer mitochondrial membrane location.
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| 9. |
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